Oral vitamin A supplementation of porcine epidemic diarrhea virus infected gilts enhances IgA and lactogenic immune protection of nursing piglets

Abstract

Vitamin A (VA) has pleiotropic effects on the immune system and is critical for mucosal immune function and intestinal lymphocyte trafficking. We hypothesized that oral VA supplementation of porcine epidemic diarrhea virus (PEDV)-infected pregnant gilts would enhance the gut-mammary gland-secretory IgA axis to boost lactogenic immunity and passive protection of nursing piglets against PEDV challenge. Gilts received daily oral retinyl acetate (30 000 IU) starting at gestation day 76 throughout lactation. At 3-4 weeks pre-partum, VA-supplemented (PEDV + VA) and non-supplemented (PEDV) gilts were PEDV or mock inoculated (mock + VA and mock, respectively). PEDV + VA gilts had decreased mean PEDV RNA shedding titers and diarrhea scores. To determine if lactogenic immunity correlated with protection, all piglets were PEDV-challenged at 3-5 days post-partum. The survival rate of PEDV + VA litters was 74.2% compared with 55.9% in PEDV litters. Mock and mock + VA litter survival rates were 5.7% and 8.3%, respectively. PEDV + VA gilts had increased PEDV IgA antibody secreting cells and PEDV IgA antibodies in serum pre-partum and IgA⁺β7⁺ (gut homing) cells in milk post piglet challenge compared with PEDV gilts. Our findings suggest that oral VA supplementation may act as an adjuvant during pregnancy, enhancing maternal IgA and lactogenic immune protection in nursing piglets.

Figure 1

Serum VA concentrations decreased immediately prior to parturition in gestating gilts. A Schematic diagram of the experimental design showing gilt PEDV infection and sample time points at post-infection day (PID) 0 [gestation day (GD) 96–97], 6–8 (GD 102–105) and 13–17 (GD 109–114) and B piglet PEDV challenge at 3–5 post-partum day (PPD) and sample time points at PPD 3–5 [PCD (post-challenge day) 0], PPD 8–14 (PCD 5–9), PPD 15–22 (PCD 12–17) and PPD 24–34 (PCD 21–29). C Pooled serum VA concentrations (ng/mL) in gilts pre and post-partum. Statistical analysis was performed using the two-way ANOVA with repeated measures and Bonferroni’s correction for multiple comparisons. Data are mean ± SEM.

Figure 2

Daily oral VA supplementation significantly decreased diarrhea in PEDV infected gilts. A Virus shedding was determined by real-time quantitative polymerase chain reaction (RT-qPCR) and expressed as log₁₀ copies/mL. B Diarrhea was determined by fecal consistency score > 1 (fecal consistency was scored as follows: 0, normal; 1, pasty/semiliquid; 2, liquid; 3, watery). Different alphabetical letters indicate significant differences among treatment groups (mean ± SEM). Statistical analysis was performed using the two-way ANOVA with repeated measures and Bonferroni’s correction for multiple comparisons. *P < 0.05.

Figure 3

Circulating PEDV specific IgA ASCs were significantly increased in PEDV + VA gilts pre-partum. A, B Peripheral blood MNCs (PBMCs) were isolated and added to PEDV ELISPOT plates to determine the PEDV specific IgA ASCs. C PBMCs were isolated and analyzed for frequencies of α4⁺β7⁺ B cells and D IgA⁺β7⁺ MNCs by flow cytometry E PEDV IgA Abs were determined by ELISA. Asterisks indicate significant differences among treatment groups at the same time point (mean ± SEM). Frequencies of α4⁺β7⁺ B cells and IgA⁺β7⁺ MNCs were normalized by dividing by post-challenge day (PCD) 0 frequencies. Statistical analysis was performed using the two-way ANOVA with repeated measures and Bonferroni’s correction for multiple comparisons. *P < 0.05 **P < 0.01 ***P < 0.001.

Figure 4

PEDV + VA litters had increased survival rates and decreased PEDV RNA shedding titers. A Kaplan–Meier survival curve of mock, mock + VA, PEDV and PEDV + VA litters at post-challenge day (PCD) 0–14. B Normalized weight gain of litters at PCD 0–15. Weights were normalized by dividing the daily weight (lbs) by PCD 0 weight (lbs). C Diarrhea was determined by fecal consistency score > 1 (fecal consistency was scored as follows: 0, normal; 1, pasty/semiliquid; 2, liquid; 3, watery). D Virus shedding was determined by real time quantitative polymerase chain reaction (RT-qPCR) and expressed as log₁₀ copies/mL. Piglet diarrhea scores and viral shedding were measured at PCD 1–7, 9, 11, 13, and 15. Different alphabetical letters indicate significant differences among treatment groups at the same time point (mean ± SEM). Statistical analysis was performed using the log-rank (Mantel-Cox) test (A) or the two-way ANOVA with repeated measures and Bonferroni’s correction for multiple comparisons (B–D).

Figure 5

PEDV + VA gilts had elevated levels of milk PEDV specific IgA ASCs and Abs, and IgA IgSCS. A Milk MNCs were isolated and added to PEDV specific and B, C total ELISPOT plates to determine the PEDV specific IgA ASCs and IgA IgSCs. D Milk MNCs were isolated and analyzed for frequencies of IgA⁺β7⁺ B cells by flow cytometry E PEDV IgA and F virus neutralizing (VN) Abs were determined by VN Ab assay, respectively. Asterisks indicate significant differences among treatment groups at the same time point (mean ± SEM). Statistical analysis was performed using a Mann–Whitney t-test (B–D) or two-way ANOVA with repeated measures and Bonferroni’s correction for multiple comparisons (A, E, F). *P < 0.05 ***P < 0.001.

Figure 6

PEDV antibody (Ab)⁺ cells in the mammary gland (MG) increased post PEDV challenge. A Evaluation of anti-PEDV Ab⁺ plasma cells in the MG pre-partum [gestation day (GD) 104–114] and post-partum [post-challenge day (PCD) 5–9] in mock, mock + VA, PEDV and PEDV + VA gilts by PEDV viral suspension sandwich immunohistochemistry (IHC) method and hematoxylin staining (300×). Right: enlarged view of MG tissue where red arrows indicate PEDV Ab⁺ cells. B Cells were quantified by averaging PEDV Ab⁺ cells from 3 to 6 microscope fields (300×) from different areas of the MG from each animal within mock, mock + VA, PEDV and PEDV + VA gilts. Statistical analysis was performed using the one-way ANOVA. ***P < 0.001.

Figure 7

Serum IgA antibodies (Abs) in PEDV (± VA) gilts increased to higher levels and at a faster rate than mock (± VA) gilts. A Serum PEDV IgA antibody secreting cells (ASCs) and B Ab responses were determined by PEDV ELISPOT and ELISA assay, respectively. C Serum PEDV virus neutralizing (VN) Ab responses were determined by VN assay. D Piglet serum circulating PEDV IgA titers were measured by ELISA. Gilts and piglet serum was collected at piglet post-challenge day (PCD) 0, 5–9, 12–17 and 21–29. Asterisks indicate significant differences among treatment groups at the same time point (mean ± SEM). Statistical analysis was performed using the two-way ANOVA with repeated measures and Bonferroni’s correction for multiple comparisons. *P < 0.05 **P < 0.01 ***P < 0.001.

Figure 8

IgA and IgG antibody secreting cells (ASCs) and α4⁺β7⁺ B cells in ileum increased in PEDV + VA gilts. A Mammary gland (MG), spleen, mesenteric lymph node (MLN) and ileum mononuclear cells (MNCs) were isolated and added to PEDV ELISPOT plates to determine the PEDV specific IgA and B IgG ASCs. C Ileum MNCs were isolated and analyzed for frequencies of α4⁺β7⁺ B cells by flow cytometry. Asterisks indicate significant differences among treatment groups at the same time point (mean ± SEM). Statistical analysis was performed a two-way ANOVA with repeated measures and Bonferroni’s correction for multiple comparisons (A, B) or a Mann–Whitney t-test (C). *P < 0.05 **P < 0.01 ***P < 0.001.